Rodent blocker

After 30 days, the pancreases were retrieved from all four groups and fixed with 4% paraformaldehyde for 6 h followed by washing and paraffin embedding. Pancreatic sections with 5- to 7-μm thickness were cut and processed for antigen retrieval. Sections were exposed to NaOH, processed further for blocking with goat serum (1%) at RT for 20 min, stained with primary antibody: polyclonal guinea pig anti-insulin, DAKO (1:100 from the stock of 26.1 g/L) overnight at 4 °C, and afterward subjected to secondary antibodies: AP-conjugated affinity-purified anti-guinea pig from Rockland, Germany (1:40 from the stock of 1 mg/mL) for 1 h at RT. Insulin was developed with a vector blue substrate kit (Vector Laboratory, Germany). Further, a rodent blocker (BIOCARE MEDICAL, Germany) was administered for 30 min before the treatment with an anti-BrdU antibody (1:100 from the stock of 262 mg/mL, DAKO, Germany) at 4 °C overnight. A mouse on mouse HRP polymer (BioCare Medical, Germany) was employed, and the BrdU-positive brown color was developed with ImmPACT™ AMEC Red Substrate. A light microscope (Leica microsystem, ICC50 HD) was utilized to capture the pictures. Morphometric analyses were standardized using blinded examiner readings.

Paraffin embedded sections of mouse brain were postfixed in neutral buffered formalin followed by antigen retrieval with Rodent Decloaker (Biocare Medical, CA, USA). Sections were blocked with avidin and biotin blocks for 20 min (Avidin Biotin Blocking Kit, Biogenex Laboratories) followed by the Rodent Blocker (Bio Care Medical, Richmond, CA, USA) and Fc receptor blocker (Innovex Biosciences) for 30 minutes at RT.
Sections were incubated with rabbit CD3 (Abcam) antibodies overnight at 4°C following secondary goat anti-rabbit antibodies (Biocare Medical, Richmond, CA, USA) for 30 min at RT. Sections were next developed with streptavidin-labeled peroxidase and Turbo DAB (Innovex Biosciences, Richmond, CA, USA) for 2–5 minutes and counterstained with hematoxylin.